A variety of diseases, both in humans and animals, is characterized by the pathogenic formation of amyloid-like fibrils or protein aggregates in neuronal tissues. A well-known and typical example of such diseases is Alzheimer's disease (AD). AD is characterized by the formation of neurofibrillar tangles and β-amyloid fibrils in the brain of AD patients. Similarly, scrapie is associated with the occurrence of scrapie-associated fibrils in brain tissue.
Another class of these diseases is characterized by an expansion of CAG repeats in certain genes. The affected proteins display a corresponding polyglutamine expansion. Said diseases are further characterized by a late onset in life and a dominant pathway of inheritance.
A typical representative of this class of diseases is Huntington's disease. Huntington's disease (HD) is an autosomal dominant progressive neurondegenerative disorder characterized by personality changes, motor impairment and subcortical dementia (Harper, 1991). It is associated with a selective neuronal cell death occurring primarily in the cortex and striatum (Vonsattel et al., 1985). The disorder is caused by a CAG/polyglutamine (polygln) repeat expansion in the first exon of a gene encoding a large ˜350 kDa protein of unknown function and designated huntingtin (HDCRG, 1993). The CAG repeat is highly polymorphic and varies from 6–39 repeats on chromosomes of unaffected individuals and 35–180 repeats on HD chromosomes (Rubinsztein et al., 1996; Sathasivam et al., 1997). The majority of adult onset cases have expansions ranging from 40–55 units, whereas expansions of 70 and above invariably cause the juvenile form of the disease. The normal and mutant forms of huntingtin have been shown to be expressed at similar levels in the central nervous system and in peripheral tissues (Trottier et al., 1995a). Within the brain, huntingtin was found predominantly in neurons and was present in cell bodies, dentrites and also in the nerve terminals. Immunohistochemistry, electron microscopy and subcellular fractionations have shown that huntingtin is primarily a cylosolic protein associated with vesicles and/or microtubules, suggesting that it plays a functional role in cytoskeletal anchoring or transport of vesicles (DiFiglia et al., 1995; Gutekunst et al., 1995; Sharp et al., 1995) Huntingtin has also been detected in the nucleus (de Rooij et al., 1996; Hoogeveen et al., 1993) suggesting that transcriptional regulation cannot be ruled out as a possible function of this protein.
In addition to HD, CAG/polygln expansions have been found in at least seven other inherited neurodegenerative disorders which include: spinal and bulbar muscular atrophy (SBMA), dentatorubral pallidoluysian atrophy (DRPLA), and the spinocerebellar ataxias (SCA) types 1, 2, 3, 6 and 7 (referenced in Bates et al. 1997). The normal and expanded size ranges are comparable with the exception of SCA6 in which the expanded alleles are smaller and the mutation is likely to act by a different route. However, in all cases the CAG repeat is located within the coding region and is translated into a stretch of polygln residues. Although the proteins harbouring the polygln sequences are unrelated and mostly of unknown function, it is likely that the mutations act through a similar mechanism. Without exception, these proteins are widely expressed and generally localized in the cytoplasm. However, despite overlapping expression patterns in brain, the neuronal cell death is relatively specific and can differ markedly (Ross, 1995), indicating that additional factors are needed to convey the specific patterns of neurodegeneration.
Several investigators have proposed that HD is caused by a toxic gain of function, which in turn is caused by abnormal protein—protein interactions related to the elongated polygln. It is possible that the binding of a protein to the polygln region could either confer a new property on huntingtin or alter its normal interactions causing selective cell death either through the specific expression patterns of the interacting protein or through the selective vulnerability of certain cells. To date, four potential huntingtin-interacting proteins have been isolated: 1 (Li et al., 1995), GAPDH (Burke et al., 1996), HIP2 (Kalchman et al., 1996) and HIP-I (Kalchman et al.,: 1997; Wanker et al., 1997). However, it has not been demonstrated whether the binding of these proteins to huntingtin is involved in the selective neuropathology. A gain of function mechanism has been supported by the identification of an antibody that specifically reacts with the pathogenic polygln expansions (Trottier et al., 1995b) This indicates that upon expansion into the pathogenic range, a polygln sequence may undergo a conformational change. Poly-L-glutamines form pleated sheets of β-strands held together by hydrogen bonds between their amides (Perutz et al., 1994). It was proposed that the expanded glutamine repeats in huntingtin may function as polar zippers, joining protein molecules together (Perutz, 1996). In the long run, this could result in the precipitation of huntingtin protein in specific neurons causing the observed selective neuronal loss. Thus, the mechanism underlying HD would be similar to scrapie, Creutzfeldt-Jakob or Alzheimer's disease, in which β-sheet secondary structures lead to the formation of toxic protein aggregates in selective neurons (Caughey and Chesebro, 1997).
Recently, strains of mice (R6) that are transgenic for the HD mutation have been generated (Mangiarini et al., 1996). In these mice exon 1 of the human HD gene carrying CAG repeat expansions of 115–156 units is expressed under the control of the human HD promoter. It has been demonstrated that the transgenic animals exhibit a progressive neurological phenotype that exhibits many of the motor and non motor features of HD. The phenotype includes a resting tremor; irregular gait; rapid, abrupt shuddering movements; stereotypic grooming movements and epileptic seizures. Coincident with the onset of the movement disorder the mice show a progressive weight loss. Neuropathological analysis has shown a reduction in brain weight (which precedes that in body weight) and the presence of neuronal intranuclear inclusions (NIIs) predating any evidence of neuronal dysfunction (Davies et al., 1997). The NIIs are immunoreactive for N-terminal huntingtin antibodies that detect the transgene protein and for ubiquitin but do not contain the endogenous mouse huntingtin. At the ultrastructural level, a solitary intranuclear inclusion appears as a roughly circular pale structure of a fine granular nature with occasional filamentous structures and devoid of a membrane. In addition, the neurons invariably have indentations of the nuclear membrane and an apparent increase in the density and clustering of nuclear pores. All three of these ultrastructural nuclear changes have previously been reported in EM studies from HD patients (Roizin et al., 1979; Roos and Bots, 1983; Tellez-Nagel et al., 1974).
Thus, a large body of data has accumulated that describes aspects of the pathology of the above-discussed diseases. However, the actual mechanisms leading to the onset of the various disease states are still unknown. Although a variety of hypotheses have been formulated in the art, it is equally unknown how the amyloid or aggregate formation is triggered or caused within affected cells or tissues. Without a detailed knowledge of the formation of said aggregates, the development of a suitable pharmaceutical composition for treating such diseases appears rather difficult. The technical problem underlying the present invention was therefore to provide means and methods suitable for the eventual elucidation of the etiology of these diseases and the development of appropriate medicines.